Chemically modified lymphokine and production thereof

ABSTRACT

The invention provides chemically modified lymphokines comprising a lymphokine moiety and at least one polyethylene glycol moiety of the formula: R.paren open-st.O--CH 2  CH 2  .paren close-st. n  wherein R is a protective group for the terminal oxygen and n is at least one, bonded directly to at least one primary amino group of the lymphokine moiety, and a method of producing the same. 
     The chemically modified lymphokines according to the invention can be produced by reacting a lymphokine with an aldehyde of the formula R.paren open-st.O--CH 2  CH 2  .paren close-st. n-1  O--CH 2  CHO wherein R and n are as defined above, in the presence of a reducing agent. 
     The chemically modified lymphokines according to the invention are useful as drugs, among others.

This is a continuation of application Ser. No. 07/407,419 filed on Sep.14, 1989, pending, which is a divisional of application Ser. No.06/919,544, filed on Oct. 14, 1986, pending, which is a file wrappercontinuation application of application Ser. No. 06/707,063, filed Mar.15, 1985 now abandoned.

Lymphokines such as interferons (hereinafter sometimes abbreviated asIFNs) and interleukin-2 (hereinafter sometimes abbreviated as IL-2) areconsidered to be of clinical value for the treatment of viral infectionsand malignancies and recent technological advances in geneticengineering have made it in principle possible to produce suchlymphokines on large scales. It is known that the clearance oflymphokines administered to the living body is in general very short. Inthe case of lymphokines derived from heterologous animals, it isanticipated that antibodies may be produced in some instances and causesevere reaction such as anaphylaxis. Therefore, technology developmentis desired which leads to delayed clearance of lymphokines used asdrugs, with their activity retained, and further to decrease in theirantigenicity. To achieve this object, chemical modification oflymphokines is a very effective means. Such chemical modification isexpected to result in delayed clearance in the living body, decreasedantigenicity and, further, increased physiological activity. From thepractical viewpoint, the significance of chemical modification oflymphokines is thus very great.

Generally, in chemically modified physiologically active proteins, amethod is required by which said proteins can be chemically modifiedwhile retaining their physiological activity. Polyethylene glycol methylether is considered to have no antigenicity and therefore is used inchemical modification of proteins. The introduction of said substanceinto proteins is generally performed by way of the intermediary ofcyanuric chloride. However, cyanuric chloride is toxic per se and thepossible toxicity of its degradation products in vivo remains open toquestion. Therefore, cyanuric chloride should be used with caution.Furthermore, the reaction involved requires a pH on the alkaline sideand therefore the above-mentioned method of modification has a drawbackin that it cannot be applied to proteins liable to inactivation underalkaline conditions.

U.S. Pat. No. 4,002,531 discloses a method of producingmonoalkylpolyethylene glycol derivatives of enzymes. However, the methoddisclosed therein, which uses sodium borohydride at pH 8.5, when appliedto lymphokines, may possibly destroy the physiological activity oflymphokines and therefore may not serve as an effective method ofproduction. Furthermore, said patent specification does not provide anysuggestion as to a means for delaying the in vivo clearance of theenzyme derivatives and such a means in otherwise unknown in the priorart.

There is also known a method of introducing a low molecular aldehydesuch as formaldehyde, acetaldehyde, benzaldehyde or pyridoxal intophysiologically active proteins in the presence of a boron-containingreducing agent [Methods in Enzymology, 47, 469 478 (1977); JapanesePatent Unexamined Publication No. 154 596/83]. However, application ofsaid method to lymphokines fails to achieve effective delay inclearance. Rather than a decrease in antigenicity, it is possible thatthe low molecular aldehyde introduced may serve as a hapten to therebyprovide said lymphokines with immunogenicity.

The present invention overcomes the above difficulties.

This is invention provides chemically modified lymphokines comprising alymphokine moiety and at least one polyethylene glycol moiety of theformula R.paren open-st.O--CH₂ --CH₂ .paren close-st._(n) (I) wherein Ris a protective group for the terminal oxygen atom and n is at leastone, bonded directly to at least one primary amino group of thelymphokine moiety and a method of producing the same.

In the present specification, the term "lymphokine" includes solublefactors released from lymphocytes and involved in cellular immunity andsubstances equivalent thereto in physiological activity.

Thus, the lymphokines may be genetically engineered products, productsderived from various animals including humans and further includesubstances similar in structure and in physiological activity thereto.

For instance, there may be mentioned various interferons [interferon-α(IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ)], IL 2, macrophagedifferentiating factor (MDF), macrophage activating factor (MAF), tissueplasminogen activator, and substances similar in structure and inphysiological activity thereto.

Examples of said substances similar in structure and in physiologicalactivity are substances having the structure of IFN-γ except for thelack of 2 to 4 amino acids at the N-terminal thereof (U.S. pat. appl.Ser. No. 685,819 claiming the priority PCT/JP84/00292), various IFN-γfragments lacking in the C terminal portion of IFN-γ (e.g. 15K species;U.S. pat. appln. Ser. No. 534,038), substances having the structure ofIL-2 except for the lack of the N-terminal amino acid thereof (EPC (laidopen) 91539) or the lack of 4 amino acids from the N-terminal portion(Japanese Patent Application 58-235638 filed Dec. 13, 1983) andsubstances having the structure of IL-2 except for the lack of one ormore constituent amino acids with or without one or more substituteamino acids in place of said missing one or ones, for example the IL-2analog containing serine in lieu of the 125th amino acid cysteine (EPC(laid open) 104798).

Preferred among such lymphokines are IFN-α, IFN-γ [consisting of 146amino acids (EPC (laid open) 0089676)], IFN-γ lacking in two N-terminalamino acids (IFN-γ d2), IFN-γ lacing in three N-terminal amino acids(IFN-γ d3), and IL-2.

The lymphokines to be used in the practice of the invention preferablyhave a molecular weight of 5,000 to 50,000, more preferably 10,000 to30,000.

The primary amino group of lymphokines includes the N-terminal α-aminogroup and the ε-amino group of the lysine residue.

Referring to the group represented by the above formula (I), theterminal oxygen-protecting group R is, for example, an alkyl or alkanoylgroup. The alkyl group is preferably an alkyl of 1 to 18 carbon atoms,more preferably a lower (C₁₋₄) alkyl, such as methyl, ethyl, propyl,i-propyl, butyl, i-butyl, sec-butyl or t-butyl. The alkanoyl group ispreferably an alkanoyl of 1 to 8 carbon atoms, more preferably a lower(C₁₋₆) alkanoyl, such as formyl, acetyl, propionyl, butyryl, i-butyrylor caproyl. The positive integer n is preferably not more than 500, morepreferably 7 to 120.

The group of formula (I) preferably has a molecular weight of not morethan 25,000, more preferably 350 to 6,000. From the viewpoints ofphysiological activity retention and clearance delaying effect, thegroup of formula (I) preferably has a molecular weight corresponding to1 to 10%, more preferably 2 to 5% of the molecular weight of thelymphokine to be modified.

The chemically modified lymphokine according to the invention consistsof a lymphokine moiety and the group of formula (I) directly bonded toat least one of the primary amino group of the lymphokine moiety.

When the N-terminal α-amino group is the only primary amino group in thelymphokine to be modified, the modified lymphokine has the group offormula (I) directly bonded to said amino group. When the lymphokine tobe modified has one or more lysine residues in its molecule, themodified lymphokine has the group of formula (I) directly bonded to somepercentage, preferably 15 to 80% (on the average), of said ε-aminogroups. In this case, the N-terminal α-amino group may have or may nothave the group of formula (I) directly bonded thereto.

The chemically modified lymphokines according to the invention can beproduced, for example, by reacting a lymphokine with the aldehyde of theformula R.paren open-st.O--CH₂ CH₂ .paren close-st._(n-1) O--CH₂ CHO(II) wherein R and n are as defined above, in the presence of a reducingagent.

As the boron-containing reducing agent to be used as conducting thereaction, there may be mentioned sodium borohydride and sodiumcyanoborohydride. Among them, more preferred is sodium cyanoborohydridefrom the viewpoint of selectivity of reaction or possibility of carryingout the reaction in the neighborhood of neutrality.

In carrying out the reaction, the aldehyde (II) is used in an amount ofabout 1 to 10,000 moles per mole of the lymphokine, and theboron-containing reducing agent is used in an amount of about 1 to 100moles per mole of the lymphokine. The degree of modification can beselected as desired by varying the mole ratio between lymphokine andaldehyde (II). The solvent to be used in carrying out the invention maybe any solvent which does not disturb the reaction and is, for example,a buffer such as a phosphate or borate buffer. An organic solvent whichdoes not inactivate lymphokines or disturb the reaction, such as a loweralkanol (e.g. methanol, ethanol, i-propanol) or acetonitrile, may beadded. The reaction may be conducted within a broad pH range of 3 to 14but is preferably perfomred at about pH 7 (pH 6.5-7.5). The reactiontemperature may be selected within a broad range of 0° to 80° C.,preferably 0° to 50° C., so as not to cause denaturation of lymphokines.A period of 0.5 to 100 hours, generally 10 to 80 hours, will besufficient for the reaction. The desired, chemically modifiedlymphokines can be obtained by purifying the reaction mixture bydialysis, salting out, ion exchange chromatography, gel filtration, highperformance liquid chromatography, electrophoresis, or the like ordinarymethod of purifying proteins. The degree of modification of the aminogroup or groups can be calculated by acid degradation followed by aminoacid analysis, for instance.

The above-mentioned aldehyde (II) can be produced from an ethyleneglycol derivative of the formula R.paren open-st.O--CH₂ CH₂ .parenclose-st._(n) OH (III) wherein R and n are as defined above, forinstance. The following method of producing the same is advantageous inthat the production of the corresponding byproduct carboxylic acid islittle.

Thus, the compound (III) is oxidized with pyridinium chlorochromate in ahaloalkane solvent such as methylene chloride or chloroform. In thiscase, pyridinium chlorochromate is used in an amount of 1 to 3 moles permole of compound (III) and the reaction is carried out at -10° to 50°C., preferably at room temperature, for 1 to 30 hours.

Treatment of compound (III) (n-1) with potassium butoxide in t-butanolfollowed by reaction with a bromoacetal and treatment with an acid suchas an organic acid (e.g. trifluoroacetic acid) or an inorganic acid(e.g. hydrochloric or sulfuric acid) can also give the correspondingaldehyde (II) which is longer in chain length by --O--CH₂ CH₂ -- thancompound (III). In this case, 10 to 30 moles, per mole of compound(III), of potassium t-butoxide is added to the above compound and, afterdissolution 3 to 15 moles, per mole of compound (III), of a bromoacetalis added, followed by reaction at 10° to 80° C. for 0.5 to 5 hours.After treatment of the reaction mixture in the conventional manner, theproduct is dissolved in a dilute aqueous solution of the above-mentionedacid, followed by heating for 5 minutes to 2 hours.

In each case, the reaction mixture can be subjected to purificationprocesses conventional in the field of chemistry, such as extraction,concentration, recrystallization, reprecipitation, chromatography and/ordistillation.

The chemically modified lymphokines according to the present inventionhave useful physiological activities similar to those of thecorresponding known, unmodified lymphokines and are useful as drugs,among other uses.

The chemically modified lymphokines according to the present inventionexhibit delay in clearance in vivo as compared with the correspondingknown, unmodified lymphokines and are low in toxicity and antigenicityand can be used safely for the same purposes and in the same manner asin the case of known lymphokines.

The chemically modified lymphokines according to the invention canusually by administered to mammals (monkey, dog, pig, rabbit, mouse,human) either orally or parenterally in the form of appropriatepharmaceutical compositions prepared by using carriers, diluents, etc.,which are known in themselves.

Thus, for instance, chemically modified IFN-α according to theinvention, when used as an antiviral agent, is recommendablyadministered to human adults once a day by intravenous injection in adose of 1×10⁴ to 1×10⁹ international units.

In the present specification, the amino acids, when referred to byabbreviations, are abbreviated according to IUPAC-IUB (Commision ofBiological Nomenclature).

The transformant Escherichia coli 294/pHITtrp1101-d2 as disclosedhereinafter in a reference example has been deposited with Institute forFermentation, Osaka (IFO) under the deposit number IFO-14350 and, sinceJun. 6, 1984, with the Fermentation Research Institute (FRI), Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry under the deposit number FERM BP-703 under Budapest Treaty.

The strain Escherichia coli DH1/pTF4 has been deposited with theInstitute for Fermentation, Osaka under the deposit number IFO-14299and, since Apr. 6, 1984, with the FRI under the deposit number FERMBP-628 under Budapest Treaty.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the clearance-delaying effect in rat plasma as disclosed inExample 1 (iv). The measurement results obtained with the chemicallymodified IFN-α according to the invention as produced in Example 1 (i)are indicated by O (enzyme immunoassay) and □ (antiviral activityassay), and the results obtained with rIFN-αA used as a control by (enzyme immunoassay) and ▪ (antiviral activity assay.

FIG. 2 shows the clearance-delaying effect in rat plasma as disclosed inExample 3 (ii). The data indicated by Δ, □ and  are the enzymeimmunoassay data for compound No. 8, compound No. 2 (Table 1) andcontrol rIFN-αA, respectively.

FIG. 3 shows the construction scheme for the expression plasmidpHITtrp1101-d2 disclosed in Reference Example 3 (i) and FIG. 4 theconstruction scheme for the expression plasmid pLC2 disclosed inReference Example 4 (i).

EXAMPLES

The following working examples and reference examples illustrate theinvention in more detail but are by no means limitative of theinvention.

EXAMPLE 1 Production of Polyethylene Glycol Methyl Ether-Modified IFN-α

(i) A 5-ml (4.8 mg as protein) portion of a solution of IFN-α (rIFN-αA)was dialyzed against 0.2M phosphate buffer (pH 7.0) and 0.15M sodiumchloride at 4° C. for 12 hours. To the dialyzate taken out, there wasadded the polyethylene-glycol methyl ether aldehyde (average molecularweight 1,900) (260 mg) obtained in Reference Example 1. Then, sodiumcyanoborohydride (140 mg) was added, and the mixture was stirred at 37°C. for 40 hours. The reaction mixture was poured into a Sephadex G-75column (3.0×43.0 cm) and developed with 25 mM ammonium acetate buffer(pH 5.0) and 0.15M sodium chloride. The eluate was collected in 5-mlportions. Eluate fractions (100-150 ml) containing the contemplatedproduct were combined. Assaying by the Lowry method using bovine serumalbumin as a standard revealed that the protein content in the combinedfractions was 84 μg/ml. Amino acid ratios in acid hydrolysate (6Nhydrochloric acid, 110° C., 24 hours) were as follows: Asp, 12.2 (12);Thr, 10.4 (10); Ser, 16.0 (14); Glu, 24.8 (26); Pro, 6.0 (5); Gly, 6.3(5); Ala, 8.6 (8); Val, 6.5 (7); Met, 4.0 (5); Ile, 7.6 (8); Leu, 21.0(21); Tyr, 5.2 (5); Phe, 9.9 (10); Lys, 6.5; His, 3.8 (3); Arg, 9.1 (9);Cys, Trp, decomposed. In view of the fact that rIFN-αA contains 11 Lysresidues, the above results led to a conclusion that about 41% of Lysresidue in interferon α had been modified as the ε-amino group with thepolyethylene glycol methyl ether (average molecular weight 1,900). Thepotency of this product as determined by the enzyme immunoassay method[Methods in Enzymology, 79, 589-595 (1981)] was 1.51×10⁷ internationalunits/mg and the antiviral activity as determined by the methoddescribed in Journal of Virology, 37, 755-758 (1981) was 0.57×10⁷international units/mg. This product (IFA-3) was submitted to aclearance test in rats as mentioned later herein.

(ii) Using 100 mg of the polyethylene glycol methyl ether aldehydeobtained in Reference Example 1 and having an average molecular weightof 750 and 100 mg of sodium cyanoborohydride, rIFN-αA was treated in thesame manner as (i) to give 30 ml of a solution of polyethylene glycolmethyl ether-modified IFN-α with a protein content of 130 μg/ml. Aminoacid ratios in acid hydrolysate (6N hydrochloric acid, 110° C., 24hours) were as follows: Asp, 12.1 (12); Thr, 10.1 (10); Ser, 13.6 (14);Glu, 26.7 (26); Pro, 5.5 (5); Gly, 5.6 (5); Ala 8.4 (8); Val, 6.7 (7);Met, 5.5 (5); Ile, 7.4 (8); Leu, 21.0 (21); Tyr, 5.1 (5); Phe, 9.6 (10);Lys, 4.7; His, 3.5 (3); Arg, 9.1 (9); Trp, 1.8 (2); Cys, decomposed. Theabove data indicate that about 57% of Lys residues had been modified atthe ε-amino group. Enzyme immunoassay performed in the same manner as(i) gave the result 5×10⁶ international units/mg, and the antiviralactivity of the product was 0.14×10⁸ international units/mg.

(iii) The produced of (i) was followed using 27 mg of the polyethyleneglycol methyl ether aldehyde and 27 mg of sodium cyanoborohydride andthere was obtained 50 ml of a polyethylene glycol methyl ether-modifiedIFN-α solution with a protein content of 45 μg/ml. Amino acid ratios inacid hydrolysate (6N hydrochloric acid, 110° C., 24 hours) gave thefollowing results: Asp, 13.6 (12); Thr, 10.4 (10); Ser, 14.9 (14); Glu,26.6 (26); Pro, 5.5 (5); Gly, 6.1 (5); Ala, 8.3 (8); Val, 6.6 (7); Met,5.2 (5); Ile, 7.4 (8); Leu, 21.0 (21); Tyr, 5.3 (5); Phe, 10.2 (10);Lys, 9.0; His, 3.6 (3); Arg, 9.1 (9); Trp, 2.3 (2); Cys, decomposed. Theabove data indicate that about 18% of Lys residues had been modified atthe ε-amino group. Enzyme immunoassay performed in the same manner as(i) gave the result 1.09×10⁸ international units/mg and the antiviralactivity of this product was 1.53×10⁸ international units/mg.

(iv) The chemically modified IFN-α (IFA-3) of the invention as obtainedabove in (i) was administered to a group of three 7-week-old female SDrats by injection into the femoral muscle in a dose of 1.274×10⁶ unitsper capita. After a prescribed period, blood was sampled from the caudalvein and the IFN-α potency in plasma was determined by the enzymeimmunoassay method and antiviral activity method described in Example 1(i). A distinct delay in clearance was observed as compared with a groupadministered unmodified interferon α (rIFN-αA) in a dose of 1.259×10⁶units per capita.

The above results are depicted in FIG. 1.

EXAMPLE 2

To 5 ml of the solution of chemically modified IFN-α (IFA-3) of theinvention as obtained in Example 1 (i), there is added 250 mg of humanserum albumin. The resulting solution is filtered through a membranefilter (pore size: 0.2 μm) and distributed into 5 vials, followed bylyophilization and storage. The contents of each vial are dissolved in 1ml of distilled water for injection just prior to use.

EXAMPLE 3 Production of Polyethylene Glycol Methyl Ether-Modified IFN-αand Alkanoyl-Polyethylene Glycol-Modified IFN-α

(i) The title compounds referred to above were synthesized by using thepolyethylene glycol methyl ether aldehyde and alkanoyl-polyethyleneglycol aldehyde obtained in Reference Example 1 and Reference Example 2,respectively, and following the procedure of Example 1. Various data foreach derivative synthesized are shown in Table 1 and amino acid analysisdata therefor in Table 2.

(ii) The chemically modified IFN-α species obtained in (i) above(compounds No. 2 and No. 8) in Table 1 were administered to 7-week-oldfemale SD rats in groups of 3 by intramuscular injection into the femurin doses of 3.12×10⁶ units and 2.66×10⁶ units, respectively. Thereafter,blood samples were collected from the caudal vein at times intervals andassayed for IFN-α potency in plasma by enzyme immunoassay. Obviouslydelayed clearance was noted as compared with the group given 3.82×10⁶units of unmodified IFN-α. These results are depicted in FIG. 2.

                  TABLE 1                                                         ______________________________________                                        Polyethylene glycol methyl ether-modified interferon α and              alkanoyl polyethylene glycol-modified interferon α                      ______________________________________                                                                     Reac-                                                                         tion  PEG aldehyde                               Compound                                                                              IFN-α                                                                            PEG aldehyde                                                                              temp. amount                                     No.     amount   (Av. mol. wt.)                                                                            °C.                                                                          (mg)                                       ______________________________________                                        1       5 ml     MeOPEG      37    252                                                (4.2 mg) (5000)            (ca. 20 times)                             2       5 ml     MeOPEG      37    124                                                (4.2 mg) (5000)            (ca. 10 times)                             3       5 ml     MeOPEG      37    61                                                 (4.2 mg) (5000)            (ca. 5 times)                              4       5 ml     MeOPEG      37    47                                                 (4.2 mg) (1900)            (ca. 10 times)                             5       5 ml     MeOPEG      4     110                                                (4.2 mg) (750)             (ca. 60 times)                             6       5 ml     MeOPEG      4     96                                                 (4.2 mg) (550)             (ca. 70 times)                             7       5 ml     MeOPEG      4     102                                                (4.2 mg) (350)             (ca. 120 times)                            8       5 ml     Acetyl PEG  4     184                                                (4.2 mg) (1540)            (ca. 50 times)                             9       5 ml     Caproyl PEG 4     120                                                (4.2 mg) (1100)            (ca. 50 times)                             ______________________________________                                                                      Reac-                                                             NaBH.sub.3 CN                                                                             tion                                            Compound                                                                              Addition of                                                                             amount      time   Content                                  No.     NaBH.sub.3 CN                                                                           (mg)        (hours)                                                                              OD 280 nm                                ______________________________________                                        1       Same time 50          18     0.139                                                      (ca. 200 times)                                             2       Same time 54          18     0.151                                                      (ca. 200 times)                                             3       Same time 52          18     0.210                                                      (ca. 200 times)                                             4       3 hrs     50          18     0.175                                            later     (ca. 200 times)                                             5       5 hrs     50          24     0.100                                            later     (ca. 200 times)                                             6       24 hrs    100         48     0.117                                            later     (ca. 400 times)                                             7       5 hrs     100         78     0.107                                            later     (ca. 400 times)                                             8       7.5 hrs   60          24     0.150                                            later     (ca. 240 times)                                             9       9 hrs     50          24     0.087                                            later     (ca. 200 times)                                             ______________________________________                                        Compound                                                                              Obtaines Yield    % Modifica-                                                                             EIA                                       No.     (ml)     (%)      tion      AVA                                       ______________________________________                                        1       30       99       31        2.02 × 10.sup.7                                                         8.63 × 10.sup.6                     2       22       79       18        1.30 × 10.sup.7                                                         5.53 × 10.sup.6                     3       20       100      3.6       5.00 × 10.sup.6                                                         1.58 × 10.sup.6                     4       17.5     73       13        3.31 × 10.sup.6                                                         --                                        5       36       84       51        2.60 × 10.sup.7                                                         --                                        6       25       70       46        4.70 × 10.sup.7                                                         --                                        7       36       91       79        1.28 × 10.sup.7                                                         2.95 × 10.sup.7                     8       25       89       40        1.77 × 10.sup.7                                                         4.27 × 10.sup.6                     9       35       73       56        2.57 × 10.sup.7                                                         --                                        ______________________________________                                         PEG: Polyethylene glycol, MeOPEG: Polyethylene glycol methyl ether, The       value in parentheses is the average molecular weight.                         NaBH.sub.3 CN: Sodium cyanoborohydride, EIA: Enzyme immounoassay, AVA:        Antiviral activity                                                       

                  TABLE 2                                                         ______________________________________                                        Amino acid analysis value                                                     ______________________________________                                        Com-                                                                          pound                                                                         No.      1      2        3    4      5    6                                   ______________________________________                                        Asp      12.8   12.7     12.5 12.5   13.4 12.9                                Thr      11.7   11.6     11.2 10.9   11.3 11.4                                Ser      15.8   16.7     15.7 15.4   17.6 15.6                                Glu      27.4   27.0     26.7 27.3   27.8 27.3                                Pro      --     5.3      5.6  5.5    5.6  5.8                                 Gly      4.9    5.0      4.6  4.6    7.1  4.6                                 Ala      8.1    8.0      8.1  7.8    8.6  7.5                                 Cys      --     --       --   --     --   --                                  Val      6.8    6.8      6.7  6.6    7.3  6.7                                 Met      3.2    4.7      4.3  4.3    4.4  4.3                                 Ile      7.7    7.7      7.7  7.6    8.0  7.6                                 Leu      21.0   21.0     21.0 21.0   21.0 21.0                                Tyr      4.3    4.5      4.6  4.6    4.9  4.6                                 Phe      9.8    9.8      9.8  9.8    9.8  9.8                                 Lys      8.6    10.3     10.6 9.6    5.4  6.1                                 His      2.7    3.0      2.7  2.7    2.9  2.8                                 Arg      8.8    8.8      9.2  8.8    9.1  8.8                                 Trp      --     --       --   --     --   --                                  ______________________________________                                                  Com-                           Theo-                                          pound                    rIFN  retical                                        No.   7      8      9                                                         α A                                                                           value                                                         ______________________________________                                                  Asp   12.2   12.5   12.8 12.6  12                                             Thr   10.9   11.6   11.3 11.6  10                                             Ser   15.4   16.8   15.6 15.6  14                                             Glu   26.1   26.3   26.4 27.6  26                                             Pro   5.5    5.7    5.7  3.7   5                                              Gly   4.5    5.3    5.4  4.6   5                                              Ala   7.3    8.3    8.4  7.8   8                                              Cys   --     --     --   --    4                                              Val   6.3    6.9    7.1  6.6   7                                              Met   4.1    4.7    4.8  3.9   5                                              Ile   7.3    7.5    7.6  7.6   8                                              Leu   21.0   21.0   21.0 21.0  21                                             Tyr   4.4    4.8    4.8  4.6   5                                              Phe   9.4    9.7    9.8  9.8   10                                             Lys   2.3    6.6    4.9  11.3  11                                             His   2.6    2.9    2.9  4.1   3                                              Arg   8.5    7.7    7.6  8.9   9                                              Trp   --     0.8    1.0  --    2                                    ______________________________________                                         --: Not detected.                                                        

EXAMPLE 4 Production of Polyethylene Glycol Methyl Ether-ModifiedInterferon-γ

(i) A 5-ml portion (5.95 mg as protein) of a solution of theinterferon-γ protein produced by the recombinant DNA technique(hereinafter abbreviated as rIFN-γ; cf. EPC laid open No. 110044) wasapplied to a Sephadex G-25 column (2.0×60.0 cm) and developed with 0.2Mphosphate buffer (pH 7.0). The eluate wad fractionated in 5-ml portions.Fractions Nos. 11-13 were combined and diluted to 100 ml with the samebuffer. Thereto was added polyethylene glycol methyl ether aldehyde(average molecular weight 750) (225 mg), followed by addition of sodiumcyanoborohydride (300 mg). The mixture was shaken at 37° C. for 72hours. The resulting precipitate was removed by centrifugation. Thesupernatant was concentrated to 10 ml using a Diaflow membrane (Amicon).The concentrate was applied to a Sephadex G-75 column (3.0×43.0 cm) anddeveloped with 25 mM ammonium acetate buffer (pH 6.0)+0.15M sodiumchloride+10 mM glutathione. The eluate was fractionated in 5-mlportions. Fractions Nos. 17-24 containing the desired product werecombined. The protein content in the combined fractions as determined bythe Bradford method using bovine serum albumin as a standard was 7.73μg/ml. The acid hydrolysate (6N hydrochloric acid, 110° C., 24 hours)gave the following amino acid analysis values: Asp, 19.6 (20); Thr, 4.7(5); Ser, 8.3 (11), Glu, 18.5 (18); Pro, 2.1 (2); Gly, 5.4 (5); Ala, 7.5(8); Val, 8.4 (8); Met, 3.7 (4); Ile, 7.1 (7); Leu, 9.7 (10), Tyr, 5.3(5); Phe, 9.7 (10); Lys, 17.6; His, 2.0 (2); Arg, 5.0 (8); Cys, Trp,decomposed. Since rIFN-γ contains 20 Lys residues, the above resultsindicate that about 12% of the Lys ε-amino groups in rIFN-γ had beenmodified by polyethylene glycol methyl ether (average molecular weight750). The product had an antiviral activity of 1.3×10⁶ internationalunits/mg. Administration of the product to rats resulted in obviousdelay in clearance in blood. On the other hand, the precipitate wasdissolved in 6M guanidine hydrochloride and dialyzed against 25 mMammonium acetate (pH 6.0)+0.15M sodium chloride+10 mM glutathione at 4°C. overnight, followed by Sephadex G-75 gel filtration in the samemanner as above. The thus-purified fraction (25 ml) had a proteincontent of 126 μg/ml and amino acid analysis of the acid hydrolysate (6Nhydrochloric acid, 110° C., 24 hours) gave the following values: Asp,20.0 (20); Thr, 5.2 (5); Ser, 9.5 (11); Glu, 27.8 (18); Pro, 2.7 (2);Gly, 14.6 (5); Ala, 8.1 (8); Val, 8.5 (8); Met, 4.3 (4); Ile, 7.2 (7);Leu, 10.2 (10); Tyr, 5.8 (5); Phe, 10.1 (10); Lys, 14.7; His, 2.0 (2);Arg, 7.3 (8); Thr, 0.7 (1); Cys, decomposed. The higher values for Gluand Gly than the theoretical are presumably due to contamination byglutathione. Since rIFN-γ contains 20 Lys ε-amino groups, the aboveresults indicate that about 26.5% of the Lys ε-amino groups in rIFN-γhad been modified by polyethylene glycol methyl ether.

(ii) Using 225 mg of polyethylene glycol methyl ether aldehyde having anaverage molecular weight of 750 and 120 mg of sodium cyanoborohydride,rIFN-γ was treated in the same manner as (i) in the presence of2-mercaptoethanol (2%) to give 30 ml of a polyethylene glycol methylether-modified rIFN-γ solution having a protein content of 236 μg/ml.Amino acid analysis of the acid hydrolysate (6N hydrochloric acid, 110°C., 24 hours) gave the following values: Asp, 20.0 (20); Thr, 5.2 (5);Ser, 9.6 (11); Glu, 33.6 (18); Pro, 1.8 (2); Gly, 19.9 (5); Ala, 8.2(8); Val, 8.9 (8); Met, 4.6 (4); Ile, 7.4 (7); Leu, 10.2 (10); Tyr, 5.9(5); Phe, 10.7 (10); Lys, 10.2; His, 2.3 (2); Arg, 7.9 (8); Trp, 0.6(1); Cys, decomposed. The higher values for Glu and Gly are presumablydue to contamination with glutathione. Since rIFN-γ contains 20 Lysε-amino groups, the above results indicate that about 50% of the Lysε-amino groups in rIFN-γ had been modified by polyethylene glycol methylether.

EXAMPLE 5 Production of Polyethylene Glycol Methyl Ether-ModifiedIFN-γd2

(i) A 5-ml portion (4.95 mg as protein) of the IFN-γd2 solution obtainedin Reference Example 3 is applied to a Sephadex G-25 column (2.0×60.0cm) and developed with 0.2M phosphate buffer (pH 7.0). The eluate isfractionated by 5 ml. Fractions Nos. 11-13 are combined and diluted to100 ml with the same buffer. To the dilution is added polyethyleneglycol methyl ether aldehyde (average molecular weight 750) (200 mg),and then sodium cyanoborohydride (300 mg). The mixtuure is shaken at 37°C. for 72 hours. The resulting precipitate is removed by centrifugation.The supernatant is concentrated to 10 ml using a Diaflow membrane(Amicon). The concentrate is applied to a Sephadex G-75 column (3.0×43.0cm) and developed with 25 mM ammonium acetate buffer (pH 6.0)+0.15Msodium chloride+10 mM glutathione. The eluate is fractionated by 5 ml,and the fractions containing modified IFN-γd2 having the polyethyleneglycol methyl ether moiety on the Lys ε-amino group in the molecule arecollected and combined. When this product is administered to rats,evident delay in clearance in blood is noted.

On the other hand, the precipitate is dissolved in 6M guanidinehydrochloride, dialyzed against 25 mM ammonium acetate buffer (pH6.0)+0.15M sodium chloride+10 mM glutathione at 4° C. overnight, andpurified by Sephadex G-75 gel filtration in the same manner as above.Thus is obtained a fraction containing modified IFN-γd2 having thepolyethylene glycol methyl ether moiety on the Lys ε-amino group in themolecule.

EXAMPLE 6 Production of Polyethylene Glycol Methyl Ether-ModifiedIFN-γd3

(i) A 5-ml (5.5 mg as protein) portion of the IFN-γd3 solution obtainedin Reference Example 4 is applied to a Sephadex G-25 column (2.0×60.0cm), followed by development with 0.2M phosphate buffer (pH 7.0). Theeluate is fractionated in 5-ml portions. Fractions Nos. 11-13 arecombined, and thereto are added polyethylene glycol methyl-etheraldehyde (average molecular weight 750) (225 mg) and then sodiumcyanoborohydride (120 mg). The mixture is shaken at 37° C. for 24 hours.The reaction mixture is applied to a Sephadex G-75 column (3.0×43.0 cm),followed by development with 25 mM ammonium acetate buffer (pH 6.0).Thus is obtained a fraction containing modified IFN-γd3 with thepolyethylene glycol methyl ether moiety on the Lys ε-amino group in themolecule. When this product is administered to rats, obvious delay inclearance in blood is observed.

EXAMPLE 7 Production of Polyethylene Glycol Methyl Ether-Modified IL-2

(i) A 5-ml (5.0 mg as protein) portion of the interleukin 2 (hereinafterabbreviated as rIL-2) obtained in Reference Example 5 was dialyzedagainst 0.2M phosphate buffer (pH 7.15) for 12 hours. To the dialyzatewas added polyethylene glycol methyl ether aldehyde (average molecularweight 750) (97 mg), and then sodium cyanoborohydride (100 mg). Themixture was stirred at 37° C. for 24 hours. The resultant precipitatewas removed by centrifugation. The supernatant was dialyzed against 5 mMammonium acetate buffer (pH 5.0) for 5 hours. The dialyzate was appliedto a Sephadex G-75 column (3.0×43.0 cm) and developed with the samesolvent system. The eluate was fractionated in 5-ml portions. Thedesired product-containing fractions Nos. 21-29 were combined. Thecombined fraction had a protein content of 25 μg/ml as determined by theBradford method using bovine serum albumin as a standard. The acidhydrolysate (6N hydrochloric acid, 110° C., 24 hours) gave the followingamino acid analysis values: Asp, 12.0 (12); Thr, 12.5 (13); Ser, 7.1(8); Gly, 18.6 (18); Pro, 5.5 (5); Gly, 2.2 (2); Ala, 5.0 (5); Val, 3.7(4); Met, 3.9 (4); Ile, 8.1 (8); Leu, 22.2 (22); Tyr, 3.0 (3); Phe, 6.0(6); Lys, 7.3; His, 3.0 (3); Arg, 3.9 (4); Cys, Trp, decomposed. SincerIL-2 contains 11 Lys residues, the above results indicate that about33.6% of the Lys ε-amino groups had been modified by polyethylene glycolmethyl ether. The IL-2 activity of the product as determined by themethod of Hinuma et al. [Biochemical and Biophysical ResearchCommunications, 109, 363-369 (1982)] which measures the growth of anIL-2-dependent mouse natural killer cell line (NKC3) with the [³H]-thymidine uptake into DNA as an index was 22,998 units/mg. When rIL-2is supposed to have an activity of 40,000 units/mg, the product isestimated to retain 57.7% of the activity. After administration to amale SD rat (5 weeks old) of this product, obvious delay in clearance inblood was noted.

REFERENCE EXAMPLE 1 Synthesis of Polyethylene Glycol Methyl-EtherAldehyde

(i) Polyethylene glycol methyl ether (5 g; average molecular weight5,000) was dissolved in methylene chloride (100 ml) and then pyridiniumchlorochromate (330 mg) was added. The mixture was stirred at roomtemperature for 12 hours. The reaction mixture was diluted two-fold withmethylene chloride and poured into a Florisil column (6×10 cm), and thecolumn was washed with methylene chloride and then with chloroform,followed by elution with methanol-chloroform (1:9). Fractions positiveto 2,4-dinitrophenylhydrazine test were combined, the solvent wasdistilled off under reduced pressure, and there was obtained acrystalline wax. Yield 1.5 g (30%). Thin layer chromatography: R_(f)=0.08 (chloroform-methanol-acetic acid=9:1:0.5, silica gel). ¹³ C-NMRspectrometry revealed an absorption due to the aldehyde group inhydrated form (--CH(OH)₂) at 96.2 ppm.

(ii) Polyethylene glycol methyl ether (10 g; average molecular weight5,000) was dissolved in tertiary-butanol (100 ml). Thereto was addedpotassium tertiary-butoxide (4.17 g), followed by addition ofbromoacetal (2.56 ml). The mixture was stirred at 40° C. for 2 hours.The tertiary-butanol was then distilled of under reduced pressure, waterwas added to the residue, and the aqueous mixture was extracted withchloroform (200 ml×2). The extract was washed with water and dried overanhydrous sodium sulfate. The chloroform was then distilled off underreduced pressure, petroleum benzine was added to the residue, and theresultant crystalline residue was collected by filtration and washedwith ether. Thus was obtained 9.5 mg (95%) of the correspondingpolyethylene glycol methyl ether diethyl acetal. A 5-g portion of theacetal was dissolved in 50 ml of 0.05M trifluoroacetic acid, treated ina boiling water bath for 30 minutes and then lyophilized, giving apolyethylene glycol methyl ether aldehyde longer in chain length by--O--CH₂ CH₂ -- than the product obtained in (i).

(iii) Polyethylene glycol methyl ether (5.7 g; average molecular weight1,900) was dissolved in methylene chloride (100 ml) and then pyridiniumchlorochromate (970 mg) was added. The mixture was stirred at roomtemperature for 12 hours, then diluted with an equal volume of methylenechloride, and poured into a Florisil column (6.0×10.0 cm). The columnwas washed with methylene chloride and then with chloroform, followed byelution with 10% methanol/chloroform. Fractions positive to2,4-dinitrophenylhydrazine test were combined. Removal of the solvent bydistillation gave a crystalline wax. Yield 1.8 g (30%). Thin layerchromatography: R_(f) =0.10 (chloroform-methanol-acetic acid=9:1:0.5,silica gel). ¹³ C-NMR spectrometry indicated the presence of anabsorption due to the aldehyde group in hydrated form (--CH(OH)₂) at96.2 ppm.

(iv) Polyethylene glycol methyl ether (19.5 g; average molecular weight1,900) was dissolved in tertiary-butanol (100 ml). Potassiumtertiary-butoxide (10.4 g) was added and then bromoacetal (6.4 ml) wasadded. The mixture was stirred at 40° C. for 2 hours. Thetertiary-butanol was then distilled off under reduced pressure. Waterwas added to the residue, followed by extraction with chloroform (200ml×2). The extract was washed with water and dried over anhydrous sodiumsulfate. The chloroform was distilled off under reduced pressure,petroleum benzine was added to the residue, and the resultantcrystalline residue was collected by filtration and washed with ether togive 8.5 g (89.5%) of acetal. A 3-g portion of the acetal was dissolvedin 0.05M trifluoroacetic acid, and the solution was treated in a boilingwater bath for 30 minutes and then lyophilized to give a polyethyleneglycol methyl ether aldehyde longer in chain length by --O--CH₂ CH₂ --than the product obtained in (iii).

(v) Polyethylene glycol methyl ether species having average molecularweights of 750, 550 and 350 were derived from to the correspondingaldehyde species by following the above procedures.

REFERENCE EXAMPLE 2 Synthesis of Alkanoyl Polyethylene-Glycol Aldehyde

(i) In 50 ml of pyridine, there was dissolved 15 g of polyethyleneglycol 1540 (Wako Pure Chemical Industries) (average molecular weight1500). To the solution was added 1.85 ml of acetic anhydride. Themixture was stirred at 40° C. for 2 hours and then at room temperaturefor 16 hours. Thereafter, the solvent was distilled off under reducedpressure. The residue was dissolved in chloroform, and the solution waswashed with water, the chloroform layer was dried over anhydrous sodiumsulfate, and the chloroform was distilled off under reduced pressure.The residue was dissolved in a small amount of chloroform, a petroleumbenzine-ether (2:1) mixture was added to the solution, and the mixturewas allowed to stand to give 14 g (90%) of a crystalline wax. A 1.4-gportion of the wax was dissolved in 50 ml of methylene chloride,followed by addition of 300 mg of pyridinium chlorochromate. Theresulting mixture was stirred at room temperature for 18 hours. Thereaction mixture was applied to a silica gel C-200 (Wako Pure ChemicalIndustries) column (3×50 cm), and the column was washed with 5%methanol-chloroform (200 ml) and eluted with 10% methanol-chloroform.Fractions positive to the 2,4-dinitrophenylhydrazine test were combined,and the solvent was distilled off under reduced pressure. A crystallinewax was obtained. Yield 580 mg (41%).

(ii) In 50 ml of methylene chloride, there was dissolved 20 g ofpolyethylene glycol 1000 (Wako Pure Chemical Ind.) (average molecularweight 1000), followed by addition of 5.15 g of n-caproyl anhydride. Themixture was stirred at 70° C. for 2 hours. Then, the solvent wasdistilled off, and the residue was purified using a silica gel C-200column (3×50 cm) and elution with ethyl acetate-methanol (4:1) to give14.9 g (60%) of an oil, which solidified upon standing in arefrigerator. The subsequent oxidation with pyridinium chlorochromate asconducted in the same manner as (i) gave the corresponding aldehyde.

REFERENCE EXAMPLE 3 Production of IFN-γd2

(i) Transformant preparation

The IFN-γ expression plasmid pHITtrp1101 [cf. EPC (laid open) No.110044, Example 2 (iii)] was digested with the restriction enzymes AvaIIand PstI, and an AvaII-PstI 1 kb DNA fragment containing the IFN-γ geneportion was isolated. The protein synthesis start codon-containingoligonucleotide adapter ##STR1## chemically synthesized by thephosphotriester method was joined to the above DNA fragment at the AvaIIcohesive end thereof using T4 DNA ligase.

The above adapted-joined gene was inserted into the DNA fragmentobtained by cleavage of the plasmid ptrp771 [cf. above-citedpublication, Example 2 (ii)] with the restriction enzymes ClaI and PstI,downstream from the trp promoter in said fragment. Thus was constructedthe expression plasmid phITtrp1101-d2 coding for the ##STR2## IFN-γpolypeptide (FIG. 3).

Escherichia coli 294 was transformed with this plasmid pHITtrp1101-d2 bythe method of Cohen et al. [Proc. Natl. Acad. Sci. U.S.A., 69, 2110(1972)] to give the transformant Escherichia coli (=E. coli)294/pHITtrp1101-d2 carrying said plasmid.

(ii) Transformant cultivation

The strain E. coli 294/pHITtrp 1101-d2 carrying the plasmid constructedin (i) above was cultivated in M9 medium containing 8 μg/ml oftetracycline, 0.4% of casamino acids and 1% of glucose at 37° C. Whenthe growth reached KU 220, 3-β-indolylacrylic acid (IAA) was added to aconcentration of 25 μg/ml. Thereafter, the cultivation was continued forfurther 4 hours. After cultivation, cells were harvested bycentrifugation and suspended in 1/10 volume of 0.05M Tris-HCl (pH 7.6)containing 10% sucrose. To the suspension, there were addedphenylmethylsulfonyl fluoride, NaCl, ethylenediaminetetraacetate (EDTA),spermidine and lysozyme to concentrations of 1 mM, 10 mM, 40 mM and 200μg/ml, respectively. After standing at 0° C. for 1 hour, the suspensionwas treated at 37° C. for 3 minutes to give a lysate.

The lysate was subjected to centrifugation at 4° C. and 20,000 rpm(Servall centrifuge, SS-34 rotor) for 30 minutes to given an IFN-γd2polypeptide-containing supernatant. This supernatant had an antiviralactivity of 2.87×10⁸ U/liter culture fluid.

(iii) Purification of IFN-γd2

In 18 ml of 0.1M Tris-hydrochloride buffer (pH 7.0) containing 7Mguanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride, therewere suspended 5.9 g of cells obtained in the same manner as (ii) aboveand stored in the frozen state. The suspension was stirred at 4° C. for1 hour and then subjected to centrifugation at 10,000 x g for 30 minutesto give 20 ml of a supernatant. This supernatant was diluted with 260 mlof a buffer (pH 7.4) comprising 137 mM sodium chloride, 2.7 mM potassiumchloride, 8.1 mM disodium phosphate and 1.5 mM monopotassium phosphate(hereinafter such buffer being referred to by the abbreviation PBS) andthe dilution was applied to an antibody column (Moγ2-11.1, column volume12 ml) at a flow rate of 1 ml/minute. The column was then washed with 60ml of 20 mM sodium phosphate buffer (pH 7.0) containing 0.5M guanidinehydrochloride and eluted with 36 ml of 20 mM sodium phosphate buffer (pH7.0) containing 2M guanidine hydrochloride to give 20 ml of anantivirally active fraction.

This 20-ml fraction was applied to a Sephacryl S-200 (Pharmacia) column(2.6×94 cm, column volume 500 ml) equilibrated in advance with 25 mMammonium acetate buffer (pH 6.0) containing 1 mMethylenediaminetetraacetate, 0.15M sodium chloride, 10 mM cysteine and2M guanidine hydrochloride, followed by eltuion with the same buffer.Thus was obtained 37 ml of the antivirally active fraction.

The ##STR3## IFN-γ polypeptide (IFN-γd2) obtained weighed 5.9 mg and hada specific activity of 1.0×10⁷ U/mg.

REFERENCE EXAMPLE 4 Production of IFN-γ d3

(i) Transformant production

The IFN-γ expression plasmid pRC23/IFI-900 [cf. Example 7 of thespecification for a patent application under EPC as laid open under No.0089676] was digested with the restriction enzymes NdeI and NcoI, and a710 bp NdeI-NcoI DNA fragment (A) containing the IFN-γ gene region wasisolated. Separately, the plasmid pRC23 was digested with therestriction enzymes BglII and EcoRI, and a 265 bp DNA fragment (B)containing the γP_(L) promoter was isolated. The fragments (A) and (B)and the chemically synthesized, protein synthesis start codon-containingoligonucleotide ##STR4## were joined together using T4 DNA ligase, withthe NdeI and EcoRI cohesive ends as the sites of joining. The DNAfragment thus obtained was joined to the plasmid pRC23/IFI-900 aftertreatment with NcoI and BglII, to thereby construct an expressionplasmid, pLC2, coding for the ##STR5## IFN-γ polypeptide (FIG. 2). Thisplasmid pLC2 was used for transforming Escherichia coli RRI (pRK248cIts) by the method of Cohen et al. [supra] to give a transformant,Escherichia coli (=E. coli) PRI (pLC2,pRK248 cIts).

(ii) Transformant cultivation

The strain E. coli RRI (pLC2,pRK248 cIts) carrying the plasmidconstructed in (i) above was shake-cultured at 35° C. in 50 ml of aliquid medium containing 1% Bactotryptone, 0.5% yeast extract, 0.5%sodium chloride and 7 μg/ml tetracycline. The culture broth wastransferred to 2.5 liters of M9 medium containing 0.5% casamino acid,0.5% glucose and 7 μg/ml tetracycline, and grown at 35° C. for 4 hoursand then at 42° C. for 3 hours. Cells were harvested by centrifugationand stored at -80° C.

(iii) Purification

In 22 ml of 0.1M Tris-hydrochloride buffer (pH 7.0) containing 7Mguanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride, therewere suspended 7.1 g of frozen cells obtained in the same manner asmentioned above in (ii). The suspension was stirred at 4° C. for 1 hourand then centrifuged at 10,000 x g for 30 minutes to give 24 ml of asupernatant. This supernatant was diluted by adding 300 ml of PBS andthe dilution was applied to an antibody column (Moγ2-11.1, columncapacity 15 ml) at a flow rate of 1 ml/minute. Thereafter, the columnwas washed with 60 ml of 20 mM sodium phosphate buffer (pH 7.0)containing 0.5M guanidine hydrochloride and then eluted with 45 ml of 20mM sodium phosphate buffer (pH 7.0) containing 2M guanidinehydrochloride, to give 25 ml of an antivirally active fraction. Thisfraction (25 ml) was applied to a Sephacryl S-200 (Pharmacia) column(2.6×94 cm; column capacity 500 ml) equilibrated in advance with 25 mMammonium acetate buffer (pH 6.0) containing 1 mMethylenediaminetetraacetic acid, 0.15M sodium chloride, 10 mM cysteineand 2M guanidine hydrochloride, and eluted with the same buffer to give40 ml of an antivirally active fraction.

The thus-obtained ##STR6## IFN-γ polypeptide IFN-γ d3 weighed 7.0 mg andhad a specific activity of 2.72×10⁷ IU/mg.

REFERENCE EXAMPLE 5 Production of Non-Glycosylated Human IL-2

(i) Transformant cultivation

E. coli DH1/pTF4 [U.S. pat. appln. Ser. No. 674,556] was inoculated into50 ml of a liquid medium (pH 7.0) containing 1% Bacto tryptone (DifcoLaboratories, USA), 0.5% Bacto yeast extract (Difco Laboratories, USA),0.5% sodium chloride and 7 μg/ml tetracycline as placed in a 250-mlErlenmeyer flask. After incubation at 37° C. overnight on a swing rotor,the culture medium was transferred to a 5-liter jar fermenter containing2.5 liters of M9 medium containing 0.5% casamino acid, 0.5% glucose and7 μg/ml tetracycline. Incubation was then conducted with aeration andstirring at 37° C. for 4 hours and, after addition of 3-β-indolylacrylicacid (25 μg/ml), for further 4 hours. Cells were harvested from thethus-obtained 2.5-liter culture broth by centrifugation, frozen at -80°C. and stored.

(ii) Extraction

The freeze-stored cells (12.1 g) obtained above were suspended uniformlyin 100 ml of an extractant (pH 7.0) containing 7M guanidinehydrochloride and 0.1M Tris.HCl, the suspension was stirred at 4° C. for1 hour and the lysate was centrifuged at 28,000 x g for 20 minutes.There was obtained 93 ml of a supernatant.

(iii) Purification of IL-2 protein

The supernatant obtained above was dialyzed against 0.01M Tris.HClbuffer (pH 8.5) and then centrifuged at 19,000 x g for 10 minutes,giving 94 ml of a dialyzate supernatnat. This dialyzate supernatant wasapplied to a DE 52 (DEAE-cellulose, Whatman, Great Britain) column (50ml in volume) equilibrated with 0.01M Tris-HCl buffer (pH 8.5) forprotein adsorption. IL-2 was eluted making a linear NaCl concentrationgradient (0-0.15M NaCl, 1 liter). The active fractions (53 ml) wereconcentrated to 4.8 ml using a YM-5 membrane (Amicon, USA) and subjectedto gel filtration using a Sephacryl S-200 (Pharmacia, Sweden) column(500 ml in volume) equibrated with 0.1M Tris.HCl (pH 8.0)-1M NaClbuffer. The active fractions (28 ml) obtained were concentrated to 2.5ml using a YM-5 membrane. The concentrate was applied to a UltraporeRPSC (Altex, USA) column for adsorption, and high performance liquidchromatography was performed using a trifluoroacetic acid-acetonitrilesystem as the eluent.

An active fraction was collected at a retention time of 39 minutes underthe following conditions: column, Ultrapore RPSC (4.6×75 mm); columntemperature, 30° C.; eluent A, 0.1% trifluoroacetic acid-99.9% water;eluent B, 0.1% trifluoroacetic acid-99.9% acetonitrile; elution program,minute 0 (68% A+32% B)--minute 25 (55% A+45% B)--minute 35 (45% A+55%B)--minute 45 (30% A+70% B)--minute 48 (100% B); elution rate, 0.8ml/min.; detection wave length, 230 nm. Thus was obtained 10 ml of asolution containing 0.53 mg of non-glycosylated human IL-2 protein[specific activity, 40,000 U/mg; activity recovery from startingmaterial, 30.6%; purity of protein, 99% (determined by densitometry)].

The following references, which are referred to for their disclosures atvarious points in this application, are incorporated herein byreference.

U.S. Pat. No. 4,002,531

Methods in Enzymology, 47, 469-478 (1977)

Japanese Patent Unexamined Publication No. 154,596/83

U.S. patent application Ser. No. 685,819

U.S. patent application Ser. No. 534,038

E.P. Patent Publication 104798

Method in Enzymology, 79, 589-595 (1981)

Journal of Virology, 37, 755-758 (1981)

E.P. Patent Publication 110044

Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)

E.P. Patent Publication 0089676

U.S. patent application Ser. No. 674,556

What is claimed is:
 1. A process of making an activated polyethyleneglycol methyl ether aldehyde for the modification of proteins, theprocess comprising the steps of:(a) dissolving a polyethylene glycolmethyl ether aldehyde in a 0.2M phosphate buffer at pH 7.0; and (b)adding sodium cyanoborohydride to the solution obtained in step (a).